Marrow-derived Mononuclear Cells Research Articles

Analysis of homing potential of marrow-derived mononuclear cells in an experimentally-induced brain stroke mouse model

Primary objective: To analyse the efficacy of bone marrow-derived mononuclear cells (MNCs) in traversing the blood–brain barrier (BBB) in an experimental model of stroke.Methods and procedures: The middle cerebral artery occlusion (MCAo) mouse model was established and behavioural and histological analysis was performed, subsequently the carboxyfluorescein diacetate (CFDA)-labelled MNCs were transplanted through the tail vein immediately after 23 hours of reperfusion. The fluorescence microscopic analysis of the brain sections was analysed in both acute and sub-acute phases of transplantation.Results: The neurological deficit was confirmed by TTC staining and contra lateral turning behaviour. After 2 and 7 days of transplantation, the CFDA-labelled MNCs were observed in the infarcted regions along the line of cortex.Conclusion: The presence of the CFDA-labelled cells in the ischaemic injured brain lesions proved homing of the implanted MNCs in the infarcted regions of the brain. The successful homing of MNCs may pave way for future clinical trials using MNC in stroke.

Bone marrow mononuclear cells protect neurons and modulate microglia in cell culture models of ischemic stroke

Although several studies have provided evidence for the therapeutic potential of bone marrow-derived mononuclear cells (MNCs) in animal models of stroke, the mechanisms underlying their benefits remain largely unknown. We have determined the neuroprotective potential of MNCs in primary neuronal cultures exposed to various injuries in vitro. Cortical neurons in culture were exposed to oxygen-glucose deprivation, hypoxia, or hydrogen peroxide, and cell death was assayed by MTT, caspase-3 activation or TUNEL labelling at 24 hrs. Cultures were randomized to cotreatment with MNC-derived supernatants or media before injury exposure. In separate experiments, macrophage or microglial cultures were exposed to lipopolypolysacharide (LPS) in the presence and absence of MNC-derived supernatants. Neuronal cultures were then exposed to conditioned media derived from activated macrophages or microglia. Cytokines from the supernantants of MNC cultures exposed to normoxia or hypoxia were also estimated by enzyme-linked immunosorbant assay (ELISA). MNC-derived supernatants attenuated neuronal death induced by OGD, hypoxia, hydrogen peroxide, and conditioned macrophage/microglial media and contain a number of trophic factors, including interleukin-10, insulin-like growth factor-1, vascular endothelial growth factor, and stromal cell-derived factor-1. MNCs provide broad neuroprotection against a variety of injuries relevant to stroke.

Bone marrow cell therapy modulates remodeling of the myocardium remnant to experimental infarction in the rat: effects on the contractility and calcium handling proteins

We evaluated the effects of bone marrow‐derived mononuclear cells (MNC) on the left ventricular (LV) function, and functional and molecular remodeling of tissue remnant to myocardial infarct (MI) in the rat. Saline (MI group) or 6×106 MNC (MI+MNC) were injected in the infarct border zone of Lewis inbred rats 48h after coronary occlusion and compared to a sham group (SO). After 6 weeks, infarct size of MI+MNC (38±2%) turned smaller than MI (44±2%), and LV dilatation and dysfunction were partially attenuated. Interestingly, developed force of isolated LV muscle samples was impaired in MI (2.6±0.4g/mm2) but fully preserved in MI+MNC (5.6±0.7) when compared to SO (5.9±0.5), in spite of sizable infarcts. Contractile responses to calcium and beta‐agonist were better in muscles from MI+MNC, as well as the post‐rest potentiation of force. Concordantly, decreases in sarcoplasmic Ca2+ATPase (46%), total (47%) and Ser16‐phosphorylated phospholamban (65%) were prevented by MNC (5, 10 and 9% respectively). While no changes were noted on α1‐Na+K+ATPase, decrease in α2 isoform due to MI was not influenced by MNC. We added evidences that MNC injection restricts post‐MI cardiac dysfunction. Also, our results indicated that this therapy preserved mechanics of myocardium remnant to infarct associated to prevention on specific calcium handling protein alterations.Supported by FAPESP and CNPq.

Differentiation of cryopreserved bone marrow-derived mononuclear cells into endothelial progenitor cells

Objective:To compare the functions of endothelial progenitor cells(EPCs) differentiated from cryopreserved and fresh bone marrow-derived mononuclear cells(MNCs).Methods: The bone marrow samples were taken from swine iliac bones.The isolated MNCs were cultured or cryopreserved at-80℃ for 3 months and then cultured again.The P1-EPCs were identifed by Dil-ac-LDL and FITC-UEA-1 double staining,immunohistochemistry and flow cytometry.The EPC pick-up rate,migration,adhesion,and proliferation abilities were compared between the cryopreserved group and the fresh group.Results: Immunohistochemisty showed that the P1-EPCs of the cryopreserved group were positive for CD133(+),CD34(+),CD31() and KDR();flow cytometry also showed they were positive for CD133([17.24±3.12]%),CD34([37.21±10.85]%),CD31([72.07±13.34]%) and KDR([89.09±16.40]%).There were no significant differences in the pick-up rates([1.1±0.078]% vs [1.03±0.061]%,P=0.054),migration rates([15±0.71]% vs [14.2±0.63]%,P=0.17),adherence rates([42.7±2.1]% vs [39.5±1.7]%,P=0.11),and proliferation abilities([25.06±2.82]×104 vs [21.64±2.34]×104,P=0.089) between EPCs of the fresh and cryopreserved groups.Conclusion: Cryopreservation has no measurable influence on the numbers and functions of EPCs differentiated from bone marrow-derived MNCs,so cryopreservation can be used to obtain sufficient homogeneous EPCs in a short period for therapy using EPCs transplantation.

Bone marrow stromal cells and bone marrow-derived mononuclear cells: Which are suitable as cell source of transplantation for mice infarct brain?

There are few studies that denote whether bone marrow stromal cells (BMSC) and bone marrow-derived mononuclear cells (MNC) show the same therapeutic effects, when directly transplanted into the infarct brain. This study therefore aimed to compare their biological properties and behaviors in the infarct brain. Mouse BMSC were harvested and cultured. Mouse MNC were obtained through centrifugation techniques. Their cell markers were analyzed with FACS analysis. The MNC (10(6) cells; n = 10) or BMSC (2 x 10(5) cells; n = 10) were stereotactically transplanted into the ipsilateral striatum of the mice subjected to permanent middle cerebral artery occlusion at 7 days after the insult. Their survival, migration, and differentiation in the infarct brain were precisely analyzed using immunohistochemistry 4 weeks after transplantation. The MNC were positive for CD34, CD45, CD90, but were negative for Sca-1. The BMSC were positive for CD90 and Sca-1. The transplanted BMSC, but not MNC, extensively migrated into the peri-infarct area. Approximately 20% of the transplanted BMSC expressed a neuronal marker, NeuN in the infarct brain, although only 1.4% of the transplanted MNC expressed NeuN. These findings strongly suggest that there are large, biological differences between MNC and BMSC as cell sources of regenerative medicine for ischemic stroke.

Bone marrow cell therapy prevents infarct expansion and improves border zone remodeling after coronary occlusion in rats

Since the cell therapy benefits for myocardial infarction are mainly related to infarct reduction by regenerating lost myocardium or increasing survival of tissues at risk, we evaluated the effects of bone marrow-derived mononuclear cells (MNC), implanted after the completion of necrosis, on infarct progression and cardiac remodeling. After 48 h of induction of myocardial infarction (MI), Lewis-inbred rats were injected with 6 × 10(6) cells (MI+MNC) or saline (MI). After six weeks, scar dimension, ventricular morphology and function were analyzed by echocardiography followed by histomorphology of the infarcted and border zones. After therapy, the relative size of the infarct was smaller in MI+MNC (37 ± 1% of the left ventricle) than in MI (43 ± 1%). While the MI group exhibited parallel elongation of the infarcted (31.6 ± 3.8% increase) and reminiscent ventricular portions (33.5 ± 3.7%), MNC therapy preserved the initial infarct length. Infarcted walls were thicker (979 ± 31 mm) in the MNC group than in the untreated group (709 ± 41 mm), also demonstrating an absence of infarct expansion. In the border zones, MNC led to increased capillary densities and capillary/myocyte ratios. The cardiac systolic function remained depressed in MI, but improved by 19 ± 5% in MI+MNC which reduced the incidence of pulmonary arterial hypertension (37.5% in MI and 6.25% in MI+MNC). MNC therapy prevented the infarct expansion and thinning related to cardiac remodeling and was associated with an improvement of border zone microcirculation: as a result, MNC therapy reduced typical MI dysfunctional repercussions.

PTK787/ZK 222584 inhibits tumor growth promoting mesenchymal stem cells: Kinase activity profiling as powerful tool in functional studies

Bone marrow (BM)-derived mesenchymal stem cells (MSCs) have been shown to favour tumor growth, suggesting the relevance of pharmaceutical inhibition of MSCs for the treatment of malignancies. We tested the effect of PTK787/ZK 222584 (PTK) on the outgrowth of MSCs from human bone marrow-derived mononuclear cells (MNCs) and the migration and tube formation capacity of MSCs in vitro. PTK dose- dependently inhibited the outgrowth of BM-MSCs from BM-MNCs (LC50 1.12 μM PTK), while hematopoietic colony formation (HCF) was only slightly hampered (13 ± 19% at 1 μM PTK, and stable at ~50% at higher concentrations of PTK). Addition of 10 μM PTK inhibited proliferation of MSCs by 74 ± 6.6% compared to control (p

Abstract 4535: Therapeutic Angiogenesis by Mononuclear Cell Implantation for Critical Digit Ischemia of Patients with Connective Tissue Diseases

Systemic sclerosis (SSc or scleroderma) is an autoimmune connective tissue disease characterized by diffuse fibrosis, degenerative changes, and microvascular abnormalities. The vasculopathy mainly affects small arteries and capillaries and causes insufficient blood flow, which leads to clinical manifestations, such as Raynaud’s syndrome, fingertip ulcers, and gangrene. Recently, implantation of bone marrow-derived mononuclear cells (MNCs) has been successfully used for therapeutic neovascularization in Buerger’s disease that is thought to be an “autoimmune” vasculitis. The purpose of this study is to determine the efficacy of autologous MNC implantation into the ischemic digits of patients with connective tissue diseases. This study was performed as a prospective, non-randamized, and multicenter clinical trial. Thirty-four patients (19 SSc, 5 SLE, 2 CREST, 2 MCTD, 4 APS, 2 PN) who had painful ischemic digits with skin ulcers were enrolled in this study. Autologous MNCs obtained from bone marrow or peripheral blood were implanted into the ischemic digits. Ischemic pain and ulcers improved remarkably after MNC implantation. In particular, SSc patients showed dramatic improvement of these parameters (18 of 19 patients, 94.7%). In patients with other types of connective tissue diseases, pain and ulcers improved in 12 of 15 patients (80.0%). No serious adverse event was observed. These results demonstrate that implantation of autologous MNCs from bone marrow or peripheral blood into ischemic digits is feasible, safe and effective for improvement of pain and skin ulcers in patients with connective tissue diseases including SSc. Thus, larger, randomized and controlled trials for this cell therapy in patients with connective tissue diseases will be warranted.

Covalent coupling of a novel TLR7 agonist to antigen elicits an effective immune response in a murine melanoma tumor model

3019 Background: Toll-like receptor (TLR) signaling triggers innate immune stimulation capable of generating an adaptive immune response under appropriate conditions. TLR agonists have shown efficacy in preclinical and clinical cancer therapies. However, one limitation to this approach has been the use of TLR agonists delivered at distances away from the target (ie-subcutaneous injections for general immune stimulation) or the inability to effectively couple antigen with adjuvant. Methods: We have synthesized a novel TLR7 agonist capable of covalent coupling to primary amines under physiologic conditions. In vitro activity of adjuvant alone or antigen-adjuvant complexes were tested for innate immune activation by utilizing bone marrow-derived murine or peripheral blood mononuclear cell-derived dendritic cells (DC) with respect to DC maturation and cytokine secretion (IL12, IL6, TGFbeta and IFNgamma). Immunocompetent syngeneic C57/b6 mice were prophylactically vaccinated with intradermal with either antigen, antigen mixed with adjuvant or antigen- adjuvant coupled complexes and challenged with B16 melanoma tumor cells expressing the cOVA transgene. Results: The effective concentration (EC50) for each compound generally followed a bell shaped distribution with higher doses being inhibitory. Maximal stimulation occurred between 10 and 1000nM. Coupled adjuvant molecules retained activity with generally lower EC50 values. Coupling 1V199 to chicken ovalbumin nearly doubled median survival from 22 to 35 days following subcutaneous tumor challenge compared with chicken ovalbumin alone. Conclusions: Covalent linkage of a TLR7 agonist to antigen stimulates DC cytokine production and protects mice from tumor challenge. Use of a soluble TLR7 agonist which retains its immune stimulating properties following coupling to antigen under physiologic conditions may be useful in the development of an in situ vaccine in solid tumor therapy. No significant financial relationships to disclose.

Intracoronary delivery of autologous bone marrow mononuclear cells radiolabeled by 18F‐fluoro‐deoxy‐glucose: Tissue distribution and impact on post‐infarct swine hearts

Intracoronary injection of the bone marrow-derived mononuclear cells (MNCs) is emerging as a potentially novel therapy for ischemic heart failure. This study was aimed at assessing the efficacy of intracoronary MNC delivery in the myocardium. The in vivo distribution and myocardial homing of intracoronarily delivered MNCs in experimental Chinese swine with acute myocardial infarction (AMI) created by occlusion of left anterior descending (LAD) coronary artery for 90 min. MNCs radiolabeled with 18F-fluoro-deoxy-glucose (18F-FDG) were delivered using a coronary catheter into the infarct-related coronary artery 1 week after AMI. Dual-nuclide single photon emission computed tomography (SPECT) revealed that 1 h after cell infusion, 6.8 +/- 1.8% of 18F-FDG-labeled MNCs occurred in the infarcted myocardium with the remaining activity found primarily in the liver and spleen. In the heart, MNCs were detected predominantly in the under-perfused myocardium. The infused cells retained in the hearts at a rate highly correlated with the under-perfused lesional sizes. Pathological examination further demonstrated that 6 weeks after infusion, compared to controls, the hearts receiving MNCs exhibited less fibrosis and inflammatory infiltrate, more viable tissue, and higher vascular density. Cardiac function was significantly improved in the MNC-infused hearts. Thus, 18F-FDG labeling and dual-nuclide SPECT imaging is capable of monitoring in vivo distribution and homing of MNCs after intracoronary infusion. MNC coronary delivery may improve cardiac function and positive ventricular remodeling in the heart with AMI.

Effect of Hypoxia on Gene Expression of Bone Marrow-Derived Mesenchymal Stem Cells and Mononuclear Cells

MSC have self-renewal and multilineage differentiation potential, including differentiation into endothelial cells and vascular smooth muscle cells. Although bone marrow-derived mononuclear cells (MNC) have been applied for therapeutic angiogenesis in ischemic tissue, little information is available regarding comparison of the molecular foundation between MNC and their MSC subpopulation, as well as their response to ischemic conditions. Thus, we investigated the gene expression profiles between MSC and MNC of rat bone marrow under normoxia and hypoxia using a microarray containing 31,099 genes. In normoxia, 2,232 (7.2%) and 2,193 genes (7.1%) were preferentially expressed more than threefold in MSC and MNC, respectively, and MSC expressed a number of genes involved in development, morphogenesis, cell adhesion, and proliferation, whereas various genes highly expressed in MNC were involved in inflammatory response and chemotaxis. Under hypoxia, 135 (0.44%) and 49 (0.16%) genes were upregulated (>threefold) in MSC and MNC, respectively, and a large number of those upregulated genes were involved in glycolysis and metabolism. Focusing on genes encoding secretory proteins, the upregulated genes in MSC under hypoxia included several molecules involved in cell proliferation and survival, such as vascular endothelial growth factor-D, placenta growth factor, pre-B-cell colony-enhancing factor 1, heparin-binding epidermal growth factor-like growth factor, and matrix metalloproteinase-9, whereas the upregulated genes in MNC under hypoxia included proinflammatory cytokines such as chemokine (C-X-C motif) ligand 2 and interleukin-1alpha. Our results may provide information on the differential molecular mechanisms regulating the properties of MSC and MNC under ischemic conditions. Disclosure of potential conflicts of interest is found at the end of this article.

Improvement of musculoskeletal function by cell-based therapy using mesenchymal stem cells with a prolonged life span

Cell transplantation has recently been attempted to improve musculoskeletal function. Many types of cells, such as embryonic stem cells, fetal cardiomyocytes, myoblasts, bone marrow hematopoietic cells, and mesenchymal stem cells (MSCs), have been transplanted to functionally restore damaged or diseased tissue in animal models, and marrow-derived mononuclear cells have been injected into ischemic limb clinically. MSCs can be a useful source of cell transplantation for several reasons:they have the ability to proliferate and differentiate into mesodermal tissues, including myocytes, they entail no ethical or immunological problems, and bone marrow aspiration is an established routine procedure. When placed in appropriate in vitro and in vivo environments, MSCs can give rise to all major mesenchymal tissues, such as bone, cartilage, muscle, and adipose tissue. Direct injection of murine and porcine MSCs into skeletal muscles has been shown to be feasible in murine models of ischemic limb. Large numbers of cells must be injected into damaged sites in ischemic limb to restore muscular function in humans, and cells need to be injected into the entire limb. Until now, however, there have been no reports of a sufficient number of differentiated human myocytes ever having been obtained to restore muscular function of ischemic limb. One of the reasons for this is that the life span of human cells in vitro is limited. Human cells reach senescence or stop cell growth after a limited number of cell replications, and the average number of hMSC population doublings (PDs) has been found to be 38, implying that it would be difficult to obtain enough cells to restore the function of ischemic limb. To resolve these problems and to establish a model of cell-based therapy, prolongation of the life span of hMSCs without affecting differentiation capability is essential.

Comparison of angiogenic potency between mesenchymal stem cells and mononuclear cells in a rat model of hindlimb ischemia

Mesenchymal stem cells (MSC) are pluripotent cells that differentiate into a variety of cells including endothelial cells and vascular smooth muscle cells. Although transplantation of bone marrow-derived mononuclear cells (MNC) has already been applied for the treatment of critical limb ischemia, little information is available regarding comparison of the angiogenic potency between MSC and MNC. Accordingly, we injected equal numbers of MSC or MNC in a rat model of hindlimb ischemia and compared their therapeutic potential. Immediately after creating hindlimb ischemia, rats were randomized to receive MSC transplantation (MSC group), MNC transplantation (MNC group), or vehicle infusion (Control group). Three weeks after transplantation, the laser Doppler perfusion index was significantly higher in the MNC group than in the Control group (0.69+/-0.1 vs. 0.57+/-0.06, P<0.01). Furthermore, there was a marked improvement in blood perfusion in the MSC group (0.81+/-0.08). Capillary density was highest in the MSC group. The number of transplanted cell-derived endothelial cells was higher in the MSC group than in the MNC group. Transplanted cell-derived vascular smooth muscle cells were detected only in the MSC group. In vitro, MSC were more tolerant to apoptotic stimulus (serum starvation and hypoxia) than MNC. MSC transplantation caused significantly greater improvement in hindlimb ischemia than MNC transplantation. Compared with MNC, MSC survived well under an ischemic environment, and differentiated into not only endothelial cells but also vascular smooth muscle cells. Thus, MSC transplantation may be a new therapeutic strategy for the treatment of severe peripheral vascular disease.

Adrenomedullin enhances therapeutic potency of bone marrow transplantation for myocardial infarction in rats

Adrenomedullin (AM), a potent vasodilator, induces angiogenesis and inhibits cell apoptosis through the phosphatidylinositol 3-kinase/Akt pathway. Transplantation of bone marrow-derived mononuclear cells (MNC) induces angiogenesis. We investigated whether infusion of AM enhances the therapeutic potency of MNC transplantation in a rat model of myocardial infarction. Immediately after coronary ligation, bone marrow-derived MNC (5 x 10(6) cells) were injected into the ischemic myocardium, followed by subcutaneous administration of 0.05 microg x kg(-1) x min(-1) AM (AM-MNC group) or saline (MNC group) for 3 days. Another two groups of rats received subcutaneous administration of AM alone (AM group) or saline (control group). Hemodynamic and histological analyses were performed 4 wk after treatment. Cardiac infarct size was significantly smaller in the MNC and AM groups than in the control group. A combination of AM infusion and MNC transplantation demonstrated a further decrease in infarct size. Left ventricular (LV) maximum change in pressure over time and LV fractional shortening were significantly improved only in the AM-MNC group. AM significantly increased capillary density in ischemic myocardium, suggesting the angiogenic potency of AM. AM infusion plus MNC transplantation demonstrated a further increase in capillary density compared with AM or MNC alone. Although MNC apoptosis was frequently observed 72 h after transplantation, AM markedly decreased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells among the transplanted MNC. In conclusion, AM enhanced the angiogenic potency of MNC transplantation and improved cardiac function in rats with myocardial infarction. This beneficial effect may be mediated partly by the angiogenic property of AM itself and by its antiapoptotic effect on MNC.

Marrow-derived CD40-positive cells are required for mice to clear Cryptosporidium parvum infection.

To clear a Cryptosporidium parvum infection, mice need CD4+ T cells, major histocompatibility complex class II, and an intact CD40-CD154 signaling pathway. CD40 is constitutively expressed on marrow-derived cells such as dendritic cells and B lymphocytes and is induced by gamma interferon (IFN-gamma) on most somatic cells. To determine whether the CD40 needed to clear a C. parvum infection has to be on marrow-derived mononuclear cells or on the epithelial cells that normally harbor the parasite, we transplanted CD40-/- mice with CD40+/- bone marrow and then infected them with C. parvum. These chimeras cleared the C. parvum infection, while CD40+/- controls transplanted with CD40-/- marrow cells remained infected. CD40 expression on marrow-derived cells therefore suffices for a C. parvum infection to be cleared, while CD40 expression on intestinal epithelial cells is not sufficient. There was no difference between the acquisition of CD69 and CD154 by mesenteric lymph node T cells of C. parvum-infected animals with intact or disrupted CD40-CD154 pathways. CD4 T cells entered the intestinal laminae propriae of C. parvum-infected animals whether or not the CD40 genes of these recipients were intact. These results suggest that, for a C. parvum infection to be cleared, CD40 is not necessary for T-cell activation but may instead contribute to an effector pathway of marrow-derived cells.